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. 2006 May 10;26(19):5265-75.
doi: 10.1523/JNEUROSCI.4680-05.2006.

Asymmetric localization of Vangl2 and Fz3 indicate novel mechanisms for planar cell polarity in mammals

Mireille Montcouquiol  1 , Nathalie Sans, David Huss, Jacob Kach, J David Dickman, Andrew Forge, Rivka A Rachel, Neal G Copeland, Nancy A Jenkins, Debora Bogani, Jennifer Murdoch, Mark E Warchol, Robert J Wenthold, Matthew W Kelley
Affiliations

Asymmetric localization of Vangl2 and Fz3 indicate novel mechanisms for planar cell polarity in mammals

Mireille Montcouquiol et al. J Neurosci. .

Erratum in

  • J Neurosci. 2006 May 31;26(22):table of contents

Abstract

Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell-cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates.

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Figures

Figure 1.
Figure 1.
Vangl2 is asymmetrical localized in the organ of Corti. A, Schematic diagram illustrating a surface view of the OC in the mammalian cochlea. Proximal-to-distal and basal-to-apical axes are indicated. The OC comprises a single row of IHCs and three rows of OHCs (O1–O3) that extend along the basal-to-apical axis. Individual hair cells are separated by specific types of supporting cells including IPh cells located between IHCs, Deiters cells (D1–D3), located between OHCs and OPs, located between OHCs in OHC1 and IPs, which create a space between the row of IHC and OHC1. Finally, the proximal edge of IHCs is contacted by a row of border cells (BC). Each HC has a stereociliary bundle (red) located on its lumenal surface. Each bundle is oriented such that its central vertex is located closest to the distal edge of the epithelium. B, Affinity-purified Vangl2 antibody recognizes a band ∼65 kDa in lysates from HEK293 transfected with an untagged Vangl2 construct (lane 1), cochlea (lane2), and brain (lane 3). C, D, Lumenal surface of the OC in the apical turn of a rat cochlea at E18.5. Vangl2 (green) is colocalized with β-catenin (red) at the membranes of all epithelial cell types in the OC. Some asymmetric localization of Vangl2 is present in IPh cells (arrows) and IHCs (asterisks). In contrast, a uniform distribution of Vangl2 is present in the undifferentiated outer hair cell region (brackets). E, F, In the more mature basal turn of the same E18.5 rat cochlea, in which overall development is more advanced by comparison with the apical turn, the asymmetry of Vangl2 is more pronounced in the IPh cells (arrowheads) but is also apparent on the proximal sides of the IHCs (asterisks), as well as in the pillar cells (white arrow) and the second row OHC (yellow arrows). G, At P1, in the basal turn of the rat cochlea, there is an accumulation of Vangl2 at the junction between the proximal edges of the hair cells and distal edges of the supporting cells (arrowheads). Vangl2 protein is noticeably absent from the lateral sides of all cells as well as from the distal edges of hair cells and the proximal edges of supporting cells. H, I, High-magnification view of the IHC region (H) and OHC region (I) in a P1 rat cochlea in single focal planes. β-Catenin labeling indicates the accumulation of Vangl2 at the exact junction between a supporting cell and a hair cell (arrows) but also between two supporting cells (asterisk). J, A similar asymmetric distribution of Vangl2 (green) is present in the basal turn of the mouse cochlea at P0. K, Cryostat section of a P0 mouse cochlea showing the apical localization of Vangl2 (green, arrowheads) in the OHCs (O1–O3) and the more extended localization in the IPs (asterisk). L, In situ hybridization with a probe for Vangl2 showing expression of mRNA (purple) in both HCs and SCs in the OC from an E18.5 mouse. The deeper purple color in the IP (asterisk) is probably a result of a relatively higher level of Vangl2 expression in these cells. M, N-cadherin (red) specifically labels IHC membranes, demonstrating a colocalization with Vangl2 (green) on the proximal (arrowheads) and distal (arrows) sides of the IHC in a P2 rat cochlea. N, An antibody against β1/β2-tubulin (red) labels the lumenal surfaces of all supporting cells, suggesting a colocalization of Vangl2 (green) at the junction of HCs (asterisks) and supporting cells (arrows). Scale bars: C (for C–F), G, J, N (for M, N), 10 μm; H (for H, I), K (for K, L), 5 μm.
Figure 2.
Figure 2.
PCP is disrupted in the vestibular system of Vangl2Lp/Lp animals. A, Scanning electronic micrograph of the surface of a utricle from a Vangl2Lp/+ animal at E18.5, illustrating the flat aspect of the tissue, compared with the two cristae (Cr) that are still attached. The surface of the utricle is covered with the stereociliary bundles of the hair cells. The striolar reversal zone is illustrated by the white curved dotted line. Hair cells located on both sides of the striola are oriented such that kinocilium on each bundle points toward the striola. See D. The red dotted line indicates mediolateral axis used to obtain hair cell polarization angles in D. B, C, High-magnification images of stereociliary bundles in utricles from Vangl2Lp/+ (B) and Vangl2Lp/Lp (C) animals. Bundle orientations are indicated by arrows based on the position of the kinocilium in each hair cell (red pseudocolor on the bundle at the extreme left edge of the image in B). D, Scatter plot of stereociliary bundle orientations along a line extending along the mediolateral axis of the utricle perpendicular to the striola (red dotted line in A). In the utricle from the Vangl2Lp/+ animal, stereociliary bundles located lateral to the striola are oriented with the kinocilium on the side of the cell closest to the striola (polarization angle of 90°). Medial to the striolar reversal, there is a 180° polarity reversal, resulting in a polarization angle of 270°. In contrast, bundle orientations in a utricle from a Vangl2Lp/Lp animal appear to be randomly distributed, demonstrating a severe PCP defect. E, Luminal surface of a WT P0 utricle labeled with β1/β2-tubulin (red) and Vangl2 (green). The yellow asterisks mark the position of the hair cells, and the arrows indicate the position of the kinocilium (red), indicating the polarity of each bundle. Vangl2 is asymmetrically expressed on the proximal side of the hair cells, opposite to the kinocilium. Supporting cell membranes also express Vangl2. Scale bars: A, 200 μm; B, C, E, 10 μm.
Figure 3.
Figure 3.
Vangl2 is absent in VanglLp/Lp mice. A, B, Lumenal surface of the OC from E18.5 WT (A) and Vangl2Lp/Lp (B) littermates. Cell boundaries are labeled with phalloidin (red), and Vangl2 expression is in green. Vangl2 expression is completely absent in the cochlea from a Vangl2Lp/Lp littermate. C, Top, Brain lysates from WT (+/+, line 1), heterozygous (Lp/+, line 2), and homozygous (Lp/Lp, line 3) VanglLp (Lp) mice were analyzed by immunoblotting. Vangl2 protein was expressed in brains from WT and heterozygotes but absent in brain from homozygotes, suggesting a degradation of the protein in the mutant. C, Bottom, HEK293 cells were cotransfected with different levels of GFP-Vangl2 (WT) or GFP-Vangl2Lp (Lp) and an empty DsRed vector. The immunoblot probed with an anti-GFP antibody reveals that the expression level of Vangl2Lp is weaker than that of Vangl2, whereas the same amount of transfected protein is present in the sample, as indicated by the levels of DsRed. D, E, Vangl2 is targeted to the membrane of transfected MDCK cells, with a strong accumulation at the junction between two transfected cells (arrows). F, G, By comparison, the Vangl2Lp construct is no longer targeted to the cell membrane (arrows) and accumulates in the cytoplasm of the cells. H–K, Lumenal surfaces of E14.5 rat cochleae, electroporated with either Vangl2 (H, I) or Vangl2Lp (J, K) and maintained for 7 d in vitro (DIV). Whereas Vangl2 (green) was strongly expressed and targeted to the cell membrane, Vangl2Lp accumulated in the cytosol of the cells, with lower levels of expression. Scale bars: B (for A, B), 10 μm; G (for D–G), K (for H–K), 20 μm.
Figure 4.
Figure 4.
Scrb1 and Vangl2 physically interact, and Scrb1 is required for Vangl2 asymmetry. A, Lumenal surface of a P1 mouse cochlea labeled with an antibody against Scrb1 (green) indicates widespread expression of the protein at the membranes of all the cells in the OC. B, Confocal Z-stack cross-section of a P1 mouse cochlea labeled as in A. Top illustrates merged image, and middle and bottom indicate individual phalloidin (red) and Scrb1 (green) channels. Scrb1 is distributed along the entire basolateral membranes of the IHCs and OHCs (numbered) as well as the IPs (asterisk). C, Immunoprecipitation of Scrb1 with Vangl2. HEK293 cells were transfected with Scrb1 and GFP-Vangl2 or GFP-Vangl2Lp constructs, and proteins were immunoprecipitated with an anti-GFP antibody. The membranes were then probed with our anti-Scrb1 antibody. The presence of a band indicates that Vangl2 and Vangl2Lp can interact with Scrb1 (lanes 5, 6), but removal of the PDZ binding domain at the C terminus of Vangl2 (Vangl2Δ4) prevents this interaction (lane 7). The band in column 2 represents endogenously expressed Scrb1 in HEK293 cells. D, Structure of mammalian Scrb1, with an LRR near the N terminus, a linker region, and four PDZ domains at the C terminus. Beneath the full structure is a graphical list of the GST constructs used in the GST pull-down assay in E. E, GST pull downs indicate that Vangl2 can interact with PDZ2, PDZ3, or PDZ4, as well as other combinations of PDZ domains from Scrb1, with the strongest interaction with PDZ2 and PDZ3. In contrast, Vangl2 does not interact with the LRR or linker domains. Mutation of Vangl2 on serine S464 leads to a strong reduction in Scrb1 binding, and mutation of the last four amino acids of Vangl2 (Vangl2Δ4) completely suppresses this binding. F, Immunolocalization of Vangl2 and Scrb1 in HEK293 cells. HEK293 cells were transfected with Scrb1 (red) and GFP-Vangl2 (green) constructs, and proteins were processed for immunocytochemistry with anti-GFP and anti-Scrb1 antibodies. The two proteins colocalize strongly when coexpressed in the cells. Scale bar, 10 μm.
Figure 5.
Figure 5.
Vangl2 asymmetry is lost in Scrb1Crc/Crc and Celsr1Crsh/Crsh mutants. A, B, Lumenal surface of the OC from an E18.5 WT (A) and Scrb1Crc/Crc littermate. In contrast with WT (A), Vangl2 (green) asymmetry is lost in cochleae from Scrb1Crc/Crc mutants (B), but the protein is still targeted to the membrane. C–F, The same image as in B but with only the green channel, at four different focal planes, beginning at the lumenal surface and advancing into the tissue by 0.62 μm in each frame. In contrast with OHCs, a persistent asymmetric localization of Vangl2 can be observed in the IPh cells and pillar cells near the lumenal surface (arrows in C), but a nearly uniform distribution of the protein is present in all cells at deeper focal planes (D–F). G–J, Lumenal surface of the OC from an E18.5 WT (G, H) and Celsr1Crsh/Crsh littermate (I, J). Vangl2 alone is illustrated in H and J. In contrast with WT, there is an overall decrease of Vangl2 (green) at the membrane and a uniform distribution of the protein in the OC from the Celsr1Crsh/Crsh animal. Scale bars: A (for A, B), I (for G–J), 10 μm.
Figure 6.
Figure 6.
Fz3 is asymmetrically localized in the OC through interactions with Vangl2. A, B, Fz3 (green) is asymmetrically distributed in a P1 rat cochlea, with a pattern similar to Vangl2. C–E, Z-stack confocal cross-section labeled as in A, in a P1 rat cochlea. Fz3 (green) is localized to the apical region of two OHCs (O2, O3) and to the IP (asterisk). F–I, In contrast with WT (F, G), Fz3 (green) asymmetry is lost in E18.5 cochleae from Vangl2Lp/Lp mice (H, I). In fact, Fz3 appears to be entirely absent from the cell membranes. J, Vangl2 immunoprecipitates with Fz3 in lysates from transfected HEK293 cells (lane 4). The blot shows the presence of two bands, corresponding to the monomeric and dimeric forms of Fz3. Scale bars: B (for A, B), H (for F–I), 10 μm.
Figure 7.
Figure 7.
Asymmetric localization of Vangl2/vang, Fz3/fz, and Scribble1 in fly wing and eye and mammalian cochlea. A, B, Localization of fz and vang in the Drosophila wing and eye. A, In the wild-type wing, a hair will form at the distal vertex on the apical surface of each cell and point distally. The actin-rich hair is believed to develop at the point of accumulation and activation of fz (green) on the distal side. vang (orange) will localize on the opposite side (proximal). In the absence of fz or vang, the asymmetry of the other protein is lost, and the hairs will form in the center of cells, pointing in the wrong direction. B, In a wild-type eye, clusters of eight photoreceptors (R1–R8) called ommatidia become polarized, before they rotate according to the equator. Here again, a specific localization of fz on the R3 side and of vang on the opposite R4 side is required for proper rotation, hence PCP. C, Lumenal view of a P0 mammalian organ of Corti. The stereociliary bundle (red) on each hair cell is located at the distal edge. Our data show that Vangl2 and Fz3 are localized opposite to the site of stereociliary bundle formation (the proximal side) of the IHCs and the OHCs (O1–O3). This accumulation of Vangl2 and Fz3 corresponds to the contact zone between a supporting cell and a hair cell and is especially strong for the outer hair cells. Our data suggest that, in fact, Vangl2 and Fz3 are located on the membranes of both types of cells. The IPh cells, as well as the IPs and Ops, seems to express Vangl2 and Fz3 both proximal and distal, whereas the Deiters cells (D1–D3) seems to have only a distal expression. Scrb1 is expressed in every cell type.
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